Team:NWU-CHINA-A/Poster

Poster: NWU-CHINA-A

The Revengers to Pathogenic Bacteria

Team leader : Yu Zhang

Student member : Jinchan Xue, Qi Gao, Ruozhou Wang, Tiancheng Wang, Xinyi Yang, Yijian Huang, Zizhan Tang, Zhou Zhang, Zhuoya Liu

Primary PI : Dai-Xu Wei

Abstract

The rising risk of severe side-effects and potential resistance of antibiotics have limited their usage. Moreover, non-biodegradable medical product loaded antibiotic is one of global pollutions. To solve these problems, our team aimed to design a medical dressing based on novel human defensins (HBDs) and polyhydroxyalkanoate (PHA) from micro-organisms. HBDs, kinds of novel antimicrobial peptide (AMP) from human, which has excellent blood compatibility and effective anti-bacteria without drug resistance and immunogenicity. In order to combine HBDs to the surface of flexible PHA film in vitro, we built a fusion protein by using a natural PHA surface binding protein (PhaP). All parts of the completely biodegradable medical dressing, including HBDs, PHA and PhaP, are designed and produced in engineering bacteria. We hope that the combination of HBDs and PHA could boost the persistent anti-bacteria and histocompatibility of adhesive bandages. Besides, implementing the concept to medical field would be our future jobs.

Inspiration

People are easy to get wounded in their daily life. The following figure shows that team members got hurt accidently during the experiments. And some special rescue professions, such as firefighters, are injured more frequently in daily training or work.

In most cases, people will choose to use adhesive bandages to dress the wounds, or use an antibiotic ointment to smear the wounds. But the abuse of antibiotics can lead to problems such as drug resistance. According to a 2019 report from US Department of Health and Human Services, more than 2.8 million antibiotic-resistant infections occurred in the U.S. each year, and more than 35,000 people died as a result.

Therefore, to protect more people from lethal bacterial infection, we intend to develop an antibacterial and completely biodegradable adhesive bandage.

Problem

1. What kind of antimicrobial peptide should be chosen？

2. How can we choose a kind of antimicrobial peptide which fits our needs best?

Our original idea was to develop a completely degradable antibacterial adhesive bandage. How do we choose the right material considering some features such as the impermeability, air permeability, biodegradability, biocompatibility, etc.

3. Once the materials and antimicrobials are selected, how do we connect them?

Idea

Now, we have the novel antibacterial material AMPs and PHA material as powerful weapons to fight with antibiotic resistance. It occurs to us that we can combine AMPs and PHA in a medical product so as to realize our goal in a large scale. We chose adhesive bandage as our primary product because it is easy to be made.

To maximize the effect of AMPs, we use PhaP (polyhydroxyalkanoate surface binding protein) to fix it on the surface of the PHA. So far, the basic structure of our small medical dressing has been determined.

Section 1 (design part)

Gene Circuit

Based on the literature and experimental principles, we constructed the following gene circuit. Between PhaP and AMP, we add a (Gly4Ser) 2 flexible linker to separate PhaP from AMP so that they can fold correctly to play their roles.

Figure 3.1.1 Gene expression route map Figure 3.1.2 Protein expression schematic

Suicide Switch

In order to prevent the engineering bacteria constructed in our laboratory from escaping to the natural environment and causing genetic contamination, we used the glucose starvation suicide switch that SZPT-CHINA team used in 2019 at the suggestion of SZPT-CHINA team. When glucose is deficient in the environment, glucose starvation induces the overexpression of autolysin gene in the T-α crp promoter(acmA).

Figure 3.1.3 Suicide Switch Scheme

The experimental cycle

Figure 3.1.4 the experiment parts

If you want to know more about our experiments, please click on our next page(experiment part).

Foreground Part

Bacterial Mucin

PHA is the material for making the central island of adhesive bandage, which has antibacterial activity and plays a major bacteriostatic role. The outer layer of the adhesive bandage is non-woven fabric. In order to connect the non-woven fabric and PHA, we chose the bacterial mucin. It consists of two types of proteins, adhesion protein and cohesive protein. Cohesion protein can be self-assembled into fibers, while adhesion protein is responsible for interfacial bonding. The combination of two types proteins forms the basis of bioadhesives.

Figure 3.1.5 Toolbox of bacterial mucin

The fibrillar structures of adhesion protein have intrinsic advantages for interfacial underwater adhesion. These advantages include tolerance to environmental deterioration, self-healing arising from self-polymerization, and large fiber surface areas. So we can use bacterial mucin to connect non-woven fabric and PHA in order to increase the water resistance of our adhesive bandage.

Section 2 (experimental part)

Select AMPs

Screening of AMPs which can be successfully expressed in E. coli from literatures. Finally we decided to use HD5, HD5d5, HBD3, DCD1L, P5, P6.2 for subsequent experiments. Among them, HD5, HD5d5, HBD3, DCD1L are human-derived defensins which are friendly to the human body and P5, P6.2 are synthetic small molecule polypeptides.

Construction of strain

We constructd plasmids and synthesized genes. The plasmids were then transformed into E. coli BL21(DE3), and transformant clones were screened.

Figure 3.2.1 Colony PCR

Proteins expression

Before the protein expression experiment, we measured the OD of the bacterial solutions to obtain the best induction time.

Figure 3.2.2 the raw data of OD Figure 3.2.3 the growth curve of E.coli with AMPs is on the left, and the growth curve of E.coli with PhaP-AMPs is on the right

Picked positive single clones, expanded the cultures, added IPTG to induce overexpression of the recombinant strains, and then used the Ultrasonic Cell Disruption System to disrupt cells under suitable conditions. Verified the amount of proteins expression by SDS-PAGE. We found PhaP-HD5, PhaP-HD5d5 are the most expressed products among them.

Figure 3.2.4 the result of SDS-PAGE

In order to prove that our products are compounds of PhaP-AMPs, we also constructed a plasmid containing only PhaP and expressed it in E.coli. According to the results of SDS-PAGE, It is proved from the side that the compounds of PhaP-AMPs are expressed in E.coli successfully.

Figure 3.2.5 the result of PhaP expression

Bacteriostatic experiment

It was noticed that PhaP-HD5d5, PhaP-HD5 showed the antibacterial effects when examined on S. aureus strain RN4220.

Figure 3.2.6 the result of bacteriostatic experiment

For safety reasons, we did not conduct bacteriostatic experiments against other bacterias, but the literature reviewed can prove that these AMPs can inhibit the growth of other bacterias[1].

References are as follows:

[1] Martínez M, Polizzotto A, Flores N, Semorile L, Maffía PC. Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6.2. Microb Pathog. 2020 Feb;139:103886. doi: 10.1016/j.micpath.2019.103886. Epub 2019 Nov 25. PMID: 31778756. Sequence and Features

PHA adhesion experiment

In order to verify the adhesion between PHA and PhaP-AMPs , we combined PhaP-AMPs with electrospinning and nanoparticles made of PHA, and proposed the composite, then tested the adhesion ability by SDS-PAGE. As we can see, PhaP-HD5d5 combines the most products with PHA，whether it is electrospinning or nanoparticles.

Figure 3.2.7 the result of PHA adhesion experiment

Summary

Based on the above experiments, we found that PhaP-HD5d5 can be highly expressed in E. coli, has a good antibacterial effect, and has the closest binding to PHA. In the end, we successfully constructed the PHA-PhaP-HD5d5 antibacterial system.

Model Section 1

Our E.coli is used to produce PhaP-AMP, a kind of fusion protein, which has antibacterial activity. For that, we simulated the process of PhaP-AMP expression under IPTG induction and analyzed the antibacterial effect quantitatively.

$$ \begin{cases} \mu =\mu _m\frac{S}{K_S+S}=\frac{1}{X}\frac{dX}{dt},t\left( P \right) \leqslant t\left( MIC \right)\\ \mu =\mu _m\frac{S}{K_S+S}e^{-K_{IP}P}=\frac{1}{X}\frac{dX}{dt},t\left( P \right) \geqslant t\left( MIC \right)\\ \frac{dP}{dt}=0,t\left( X \right) \leqslant t\left( Xa \right)\\ \frac{dP}{dt}=\beta X,t\left( X \right) \geqslant t\left( Xa \right)\\ -\frac{dS}{dt}=\frac{1}{Y_{X/S}^{*}}\frac{dX}{dt}+\frac{1}{Y_{P/S}}\frac{dP}{dt}+mX\\ \end{cases} $$

Figure 4.1.1 Quality of cell or substrate changes over time in 10 hours

Through the simulation of modeling, we determined that the best induction time for the experimental design was 3 hours after culture.

Our simulation result also showed that there was no obvious inhibition to the engineering bacteria in the production, and the main factor limiting the yield was the substrate concentration.

Compared with batch culture, continuous culture may be more suitable for our project.

Through simulation, we found that when the feeding rate was 0.1 mL/s, the yield in the reactor was higher and the fermentation could last for a long time.

Figure 4.1.2 Rate of feed is 0.1 mL/s in reactor of 1.0 L

Model Section 2

We used the following equations to describe the relationship between area of the inhibition zone and concentration of AMP. And the result was in line with our expectation, namely, the antibacterial effect is proportional to the concentration of AMP.

$$ \begin{cases} S_0=\pi \left( \frac{l}{2} \right) ^2=\frac{\pi}{4}l^2\\ n_{bac}=c_0S_0=\frac{\pi c_0}{4}l^2\\ v_0c_x=c_{MIC}S_0+n_{bac}\alpha\\ \end{cases} $$

Figure 4.2.1 Diameter of inhibition zone varies with AMP concentration

Patent

We have made a model of adhesive bandage in the laboratory, which is similar to the adhesive bandages on the market in appearance, but different from ordinary adhesive bandages, it has good antibacterial ability. The antibacterial system of PHA-PhaP-AMP gives it a broader prospect.

Figure 5.1 the model of adhesive bandage

While conducting the project feasibility test in the laboratory, we are also working hard to gain market recognition. At present, the patent of our project is under application and has been professionally recognized. We are looking forward to putting it on the market as soon as possible.

Figure 5.2 the payment notice for patent

Attribution

Professor Dai-Xu Wei provided us with comprehensive guidance.

Teacher Mingqin Fan and Jiawei Wu provided us with logistical help.

Professor Lin Chen, Shiwei Wang, Hongmin Li, Feng Guan and Haihua Liang helped us when our experiment was in trouble.

Xiaohong Zhao, Xueliang Peng, Hailun Gong and Junjie Zhu are our experimental advisors.Zhuolin Liu is our modeling advisor.

Jiabao Li is an ambassador of CCiC who provided us with zoom account to help us prepare for Shaanxi iGEM Meetup.

Miss Yang Li, Dr. Liang Zhao and Professor Jing Han were interviewed by us and provided valuable suggestions for us.

We also interviewed two firefighters and a doctor. Captain Fan and Director Su of China Fire And Rescue gave us advice from different angles. Dr. Qiuli Fan introduced the pollution of medical waste to us and affirmed our project.

Team Members

Pharmacy research

Public expectations for future adhesive bandage: Most people's requirements for future adhesive bandage are mainly reflected in the three aspects of outer packaging, comfort and antibacterial properties.

①The color of the adhesive bandage should not be too strange for the outer packing. Others suggested that it would be better to make them transparent or sell by classification by skin tone.

②For comfort, the sticky part and adhesive bandage of the skin is extremely troubling for patients with latex allergies. In addition, it is also an important demand for consumers to consider both waterproof and breathability features.

③As far as bacteriostasis is concerned, most of the bandages on the market lack bacteriostasis function.

Questionnaire

After the pharmacy research, we made product improvements according to the needs of the public. In order to hear more voices, we combined with our products to do an electronic version of the questionnaire. As of October 25, we had received a total of 528 public feedback.

The results of the questionnaire showed that most people wanted a new adhesive bandage that avoids the use of antibiotics and is biodegradable. But they do not know much about PHA and antimicrobial peptides.

Interview

We interviewed firefighters, professors, a manager of biotechnology company, athletes and a doctor.

Prof. Han: Helped us optimize protein purification and increase the concentration of protein expression.

Firefighters: They said they often use adhesive bandages to deal with small wounds in their daily attendance. Besides, they often see adhesive bandages thrown away everywhere.

Manager, Ms. Li: She thought PHA is a kind of flexible material, which can achieve full biodegradation, has broad application prospects, and is of great significance to environmental protection.

Dr. Zhao: He said antibacterial peptide has a good development prospect and is an ideal raw material to replace antibiotics.

Athletes and a Doctor: After watching our presentation, they were willing to accept this new type of adhesive bandage, and hoped to promote it as soon as possible.

Acknowledgements and Sponsors

Sponsors

References

[1] Zelić B, Vasić-Racki D, Wandrey C, Takors R. Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor. Bioprocess Biosyst Eng. 2004 Jul;26(4):249-58. doi: 10.1007/s00449-004-0358-0. Epub 2004 Apr 15. PMID: 15085423.

[2] Zhao H , Wei H , Liu X , et al. Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila[J]. entific Reports, 2016, 6(1):39424.

[3] Wanniarachchi YA, Kaczmarek P, Wan A, Nolan EM. Human defensin 5 disulfide array mutants: disulfide bond deletion attenuates antibacterial activity against Staphylococcus aureus. Biochemistry. 2011 Sep 20;50(37):8005-17. doi: 10.1021/bi201043j. Epub 2011 Aug 23. PMID: 21861459; PMCID: PMC3179267.

[4] Faccone D, Veliz O, Corso A, Noguera M, Martínez M, Payes C, Semorile L, Maffía PC. Antimicrobial activity of de novo designed cationic peptides against multi-resistant clinical isolates. Eur J Med Chem. 2014 Jan;71:31-5. doi: 10.1016/j.ejmech.2013.10.065. Epub 2013 Nov 1. PMID: 24269514.

[5] Hollmann A, Martínez M, Noguera ME, Augusto MT, Disalvo A, Santos NC, Semorile L, Maffía PC. Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides. Colloids Surf B Biointerfaces. 2016 May 1;141:528-536. doi: 10.1016/j.colsurfb.2016.02.003. Epub 2016 Feb 4. PMID: 26896660.

[6] Xue Q, Liu XB, Lao YH, Wu LP, Wang D, Zuo ZQ, Chen JY, Hou J, Bei YY, Wu XF, Leong KW, Xiang H, Han J. Anti-infective biomaterials with surface-decorated tachyplesin I. Biomaterials. 2018 Sep;178:351-362. doi: 10.1016/j.biomaterials.2018.05.008. Epub 2018 May 9. PMID: 29778319.

[7] Faccone D, Veliz O, Corso A, Noguera M, Martínez M, Payes C, Semorile L, Maffía PC. Antimicrobial activity of de novo designed cationic peptides against multi-resistant clinical isolates. Eur J Med Chem. 2014 Jan;71:31-5. doi: 10.1016/j.ejmech.2013.10.065. Epub 2013 Nov 1. PMID: 24269514.

[8] Maturana P, Martinez M, Noguera ME, Santos NC, Disalvo EA, Semorile L, Maffia PC, Hollmann A. Lipid selectivity in novel antimicrobial peptides: Implication on antimicrobial and hemolytic activity. Colloids Surf B Biointerfaces. 2017 May 1;153:152-159. doi: 10.1016/j.colsurfb.2017.02.003. Epub 2017 Feb 13. PMID: 28236791.

[9] Martínez M, Polizzotto A, Flores N, Semorile L, Maffía PC. Antibacterial, anti-biofilm and in vivo activities of the antimicrobial peptides P5 and P6.2. Microb Pathog. 2020 Feb;139:103886. doi: 10.1016/j.micpath.2019.103886. Epub 2019 Nov 25. PMID: 31778756.

[10] Su Z, Li P, Wu B, Ma H, Wang Y, Liu G, Zeng H, Li Z, Wei X. PHBVHHx scaffolds loaded with umbilical cord-derived mesenchymal stem cells or hepatocyte-like cells differentiated from these cells for liver tissue engineering. Mater Sci Eng C Mater Biol Appl. 2014 Dec;45:374-82. doi: 10.1016/j.msec.2014.09.022. Epub 2014 Sep 16. PMID: 25491842.

[11] Zhao H, Wei H, Liu X, Yao Z, Xu M, Wei D, Wang J, Wang X, Chen GQ. Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila. Sci Rep. 2016 Dec 23;6:39424. doi: 10.1038/srep39424. PMID: 28009010; PMCID: PMC5180188.

[12] Böffert R, Businger R, Preiß H, Ehmann D, Truffault V, Simon C, Ruetalo N, Hamprecht K, Müller P, Wehkamp J, Schindler M. The human α-defensin-derived peptide HD5(1-9) inhibits cellular attachment and entry of human cytomegalovirus. Antiviral Res. 2020 May;177:104779. doi: 10.1016/j.antiviral.2020.104779. Epub 2020 Mar 21. PMID: 32209394.

[13] Dong Y, Li P, Chen CB, Wang ZH, Ma P, Chen GQ. The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD. Biomaterials. 2010 Dec;31(34):8921-30. doi: 10.1016/j.biomaterials.2010.08.001. Epub 2010 Aug 21. PMID: 20728212.

[14] Xie H, Li J, Li L, Dong Y, Chen GQ, Chen KC. Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins. Acta Biomater. 2013 Aug;9(8):7845-54. doi: 10.1016/j.actbio.2013.04.038. Epub 2013 Apr 29. PMID: 23639778.